Imaging dopamine receptors in the human brain by positron tomography.

Neurotransmitter receptors may be involved in a number of neuropsychiatric disease states. The ligand 3-N-[11C]methylspiperone, which preferentially binds to dopamine receptors in vivo, was used to image the receptors by positron emission tomography scanning in baboons and in humans. This technique holds promise for noninvasive clinical studies of dopamine receptors in humans.

Positron emission tomography neuroreceptor imaging as a tool in drug discovery, research and development.

Improved communication and cooperation between research-driven drug companies and academic positron emission tomography (PET) centers, coupled with improvements in PET camera resolution, the availability of small animal PET cameras and a growing list of neuroreceptor-specific PET tracers, have all contributed to a substantial increase in the use and value of PET as a tool in central nervous system drug discovery and development.

Non-invasive radiotracer imaging as a tool for drug development.

Non-Invasive Radiotracer Imaging (NIRI) uses either short-lived positron-emitting isotopes, such as 11C and 18F, for Positron Emis ion Tomography (PET) or single photon emitting nuclides, e.g., 123I, which provide images using planar imaging or Single-Photon Emission Computed Tomography (SPECT). These high-resolution imaging modalities provide anatomical distribution and localization of radiolabeled drugs, which can be used to generate real time receptor occupancy and off-rate studies in humans. This can be accomplished by either isotopically labeling a potential new drug (usually with 11C), or indirectly by studying how the unlabelled drug inhibits specific radioligand binding in vivo. Competitive blockade studies can be accomplished using a radiolabeled analogue which binds to the site of interest, rather than a radiolabeled version of the potential drug. Imaging, particularly PET imaging, can be used to demonstrate the effect of a drug through a biochemical marker of processes such as glucose metabolism or blood flow. NIRI as a development tool in the pharmaceutical industry is gaining increased acceptance as its unique ability to provide such critical information in human subjects is recognized. This section will review recent examples that illustrate the utility of NIRI, principally PET, in drug development, and the potential of imaging advances in the development of cancer drugs and gene therapy. Finally, we provide a brief overview of the design of new radiotracers for novel targets.

A new electrophoretic method for determining ligand: technetium stoichiometry in carrier free 99mTc-radiopharmaceuticals.

An electrophoretic procedure is outlined for the determination of the number of ligands bound to technetium-99m radiopharmaceuticals. The approach involves use of ligands that will complex technetium in a similar fashion but that differ in charge. This approach was applied experimentally to dimercapto ligands in which the ligating sulfur atoms are separated by a flexible three-carbon chain (1,3-dimercapto compounds). Two such ligands studied are 1,3-dimercaptopropane (DMP) and dihydrothioctic acid (DHTA). The Tc compound of DHTA migrates much farther on electrophoresis than the Tc complex of DMP. However, when TcO4- is reduced by SnCl2 or NaBH4 in the presence of equimolar quantities of DHTA and DMP, a new compound is formed being twice as abundant as either the TcDMP or the TcDHTA compound and migrating an intermediate distance. The formation of this new complex and the 1:2:1 distribution indicates that two 1,3-dimercapto compounds are attached to the Tc-center in all three compounds.

In vivo labeling of angiotensin II receptors with a carbon-11-labeled selective nonpeptide antagonist.

UNLABELLED: Angiotensin II (ANG II) initiates a variety of physiological effects by binding to high affinity receptors. Two ANG II receptor subtypes, AT1 and AT2, have recently been identified. This study was undertaken to evaluate [11C]L-159,884, an AT1 subtype selective nonpeptide antagonist, as a potential PET tracer. METHODS: Carbon-11-L-159,884 was prepared by alkylation of the nor precursor with [11C]methyliodide and was studied for its in vivo binding characteristics, biodistribution and kinetics in mice. The effects of PD-123319, an AT2-selective ANGII antagonist, as well as those of alpha- and beta-adrenergic drugs on [11C]L-159,884 binding were investigated also. RESULTS: Administration of the AT1 antagonists resulted in dose-dependent inhibition of [11C]L-159,884 binding in the kidneys, the organ with the highest density of AT1 receptors. Inhibition was also observed in the lungs and the heart. Adrenergic drugs did not influence [11C]L-159,884 binding to AT1 receptors. Kinetic studies showed rapid tracer uptake in the liver, kidneys, lungs and heart. Excretion of the radioactivity occurred primarily through the intestinal tract (> 20% in 90 min), with less than 8% excreted through the urine. CONCLUSION: The results suggest that [11C]L-159,884 binds in vivo to AT1 receptors in mouse kidneys, lungs and heart. This radiotracer appears to be a promising candidate for studying ANG II receptors in vivo by PET.

Comparison of technetium-99m aminoalkyl diaminodithiol (DADT) analogs as potential brain blood flow imaging agents.

N-ethyl piperidinyl diaminodithiol (NEP-DADT), complexed with 99mTc has been developed as an agent for the measurement of brain blood flow using SPECT. Studies in patients have shown that 99mTc NEP-DADT enters rapidly into the brain, but also clears rapidly (t1/2 = 17 min). In this study nine new aminoalkyl DADT derivatives were synthesized, labeled with 99mTc and tested in mice with the aim of developing an agent with increased retention in the brain. In addition, relationships between chemical properties of the derivatives and their in vivo localization were investigated. The results were as follows: (a) the R-group and its isomeric configuration has a profound influence on the biodistribution; (b) 99mTc aminoalkyl DADT derivatives with apparent pKa values of greater than 6.9 show poor brain uptake (less than 0.40% dose at 5 min); (c) lengthening of the chain between the DADT moiety and the amino-R group from ethyl to hexyl generally increases the apparent pKa and consequently lowers brain uptake; (d) a correlation (r = 0.71) exists between initial brain uptake and the octanol-buffer partition coefficient; (e) 99mTc-4'-methyl NEP-DADT has the highest partition coefficient, relatively high uptake, and longest retention in the mouse brain. This complex has characteristics suited for brain blood flow measurements.

4-[125I] iodophenyltrimethylammonium ion, an iodinated acetylcholinesterase inhibitor with potential as a myocardial imaging agent.

4-[125I]Iodophenyltrimethylammonium acetate (4-I-125 PTMA) was prepared by chloramine-T iodination of N,N-dimethylaniline and subsequent methylation with methyliodide. Purification by thin layer chromatography afforded a product whose specific activity approached the theoretical carrier-free level. Biodistribution studies in normal ICR mice showed a significant accumulation of 4-I-125 PTMA in the heart tissue, with heart-to-blood ratios of 12.5, 10.4, 7.8, 3.4, 3.4, and 3.3 at 1, 5, 10, 30, 60, and 120 min, respectively. Initial uptake in the heart was greater than 26% of the injected dose per gram. Twenty-five percent of the activity was excreted unchanged by the kidneys during the first 5 min. Less than half of the injected activity was retained in mice at 120 min.

(3-N-[11C]methyl)spiperone, a ligand binding to dopamine receptors: radiochemical synthesis and biodistribution studies in mice.

Carbon-11-labeled 3-N-methylspiperone, a positron-emitting dopamine-receptor antagonist with potential for use in positron emission tomography studies of human neurotransmitter receptors, was synthesized from 11CO2 in 40 min, with a radiochemical yield of approximately 20-40%. The specific activity of the (3-N-[11C]methyl)-spiperone was determined by ultraviolet spectroscopy to be approximately 270 mCi/mumol at the end of synthesis. In in vitro binding experiments, the Ki for 3-N-methylspiperone was found to be approximately 250 pM (against H-3 spiperone). The brain-to-blood ratios in normal ICR mice were 2.8 or greater at the times studied, and the striatum-to-cerebellum ratio at 60 min after injection was 20:1.

Investigation of angiotensin II/AT1 receptors with carbon-11-L-159,884: a selective AT1 antagonist.

UNLABELLED: Antagonists of the angiotensin II AT1 receptor subtype have been recently introduced for treatment of arterial hypertension and for pharmacological studies of these receptors. The purpose of this work was to label such an antagonist with 11C and test the applicability of the radioligand for PET studies. METHODS: The potent and selective nonpeptide AT1 antagonist L-159,884 was labeled with 11C and injected intravenously into six dogs. Renal accumulation and kinetics of the radioligand were imaged with PET at baseline and after receptor blockade with 1 mg/kg MK-996. Time-activity curves were derived from the renal cortex and were analyzed by the Gjedde-Patlak plot to obtain the influx rate constant of the radioligand. RESULTS: There was selective radioligand binding in the kidneys, mainly located in the cortex. Within the time interval between 95 and 115 min postinjection, the radioactivity retained in the kidneys was 109 +/- 27 and 42 +/- 4 nCi/ml/mCi of the injected dose for the control and inhibition studies, respectively. The influx rate constant of the radioligand decreased from a baseline of 0.0298 +/- 0.0156 to a post-MK-996 value of 0.0098 +/- 0.0052. CONCLUSION: These results demonstrate distinct binding of 11C-L-159,884 in the renal cortex with a specific binding component suitable for quantitative PET imaging of angiotensin II/AT1 receptors.

In vivo labeling of endothelin receptors with [(11)C]L-753,037: studies in mice and a dog.

UNLABELLED: Endothelin (ET) is a potent mammalian vasoconstrictive peptide and a pressor agent. Its 3 isoforms, ET-1, ET-2, and ET-3, mediate several physiologic actions in several organ systems, binding to 2 major receptor subtypes: ET(A) and ET(B). This study was undertaken to evaluate [(11)C]L-753,037 [(+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxy-propyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno [1,2-beta]pyridine-6-carboxylate), a new mixed ET receptor A and B antagonist, as a tracer for in vivo labeling of ET receptors in mice and a dog. METHODS: [(11)C]L-753,037 was synthesized, purified, and formulated from a normethyl precursor, L-843,974, and [(11)C]H(3)I. The tracer was studied for its in vivo kinetics, biodistribution, and ET receptor binding characteristics in mice. In the dog, PET imaging was performed to evaluate binding of [(11)C]L-753,037 to ET receptors in the heart. Specificity of binding was studied in the heart with the selective ET(A) antagonist L-753,164. RESULTS: Kinetic studies in mice showed highest tracer uptake at 5 min after injection in liver (25.0 percentage injected dose per gram [%ID/g]), kidneys (18.7 %ID/g), lungs (15.2 %ID/g), and heart (5.6 %ID/g). Initial high uptake in liver, lungs, and kidneys was followed by rapid washout during the next 10 min and a very slow clearance during the time of observation (2 h after injection). By contrast, the radioactivity in the heart remained constant over 2 h. Administration of both ET(A) (L-753,164) and mixed ET(A)/ET(B) (L-753,137) receptor antagonists resulted in dose-dependent inhibition of [(11)C]L-753,037 binding in mouse heart, lungs, and kidneys but not in the liver. Radioactivity in the brain was very low, indicating that the tracer does not cross the blood-brain barrier. In the dog, a dynamic PET study of the heart showed high tracer accumulation at 55-95 min after injection. Injection of L-753,164 at 30 min before [(11)C]L-753,037 administration led to a significant reduction in tracer binding. [(11)C]methyl triphenyl phosphonium was used as a tracer for reference images of the dog heart muscle. CONCLUSION: The results suggest that [(11)C]L-753,037 binds to ET receptors in vivo and is, therefore, a promising candidate for investigation of these receptors and their occupancy by ET receptor antagonists using PET.

Design, preparation, and biodistribution of a technetium-99m triaminedithiol complex to assess regional cerebral blood flow.

A new ligand (N-piperidinylethyl-DADT, 5) has been prepared which forms two complexes with 99mTc when stannous chloride is used as a reducing agent for [99mTc] pertechnetate. Biodistribution studies of one of the complexes in mice showed that 2.2% of the injected dose of the tracer was in the brain at 5 min postintravenous injection with 0.53% of the dose remaining in the brain at 30 min postinjection. Brain-to-blood ratios at these times were 5.3 and 3.0, respectively. Biodistribution studies of the other complex showed similar behavior with a slightly lower initial uptake by and faster clearance from the brain. Imaging studies of the more promising of the two complexes were conducted in a monkey and a baboon. In both cases, rapid uptake of the tracer in the brain was observed and clear brain images were obtained. Time-activity curves showed peak uptake in the brain at approximately 5 to 7 min postintravenous injection followed by a plateau of about 11 min. The half-lives for clearance of the tracer from the brains of the monkey and baboon were found to be 63 and 58 min, respectively. These results suggest that this tracer may be useful for brain imaging in humans.

Characterization of specific binding of [125I]L-762,459, a selective alpha1A-adrenoceptor radioligand to rat and human tissues.

L-762,459 ((+/-)1-(3-¿[5-carbamoyl-2-2-[(4-hydroxy-3-iodobenzimidoyl)-amino] -ethoxy-methy¿-6-methyl-4-(4-nitropheny)-1,4-dihydropyridine -3-carbonyl]-amino¿-propyl)-4-phenyl-1-piperidine-4-carboxylic acid methyl ester), an analog of a series of dihydropyridines previously reported to be selective alpha1A-adrenoceptor subtype antagonists was found to have alpha1A-adrenoceptor subtype selectivity (Ki (nM), la = 1.3, lb = 240, Id = 280). Specific [125I]L-762,459 binding was detected in rat cerebral cortex, hippocampus, vas deferens, kidney, heart and prostate tissues known to contain the alpha1A-adrenoceptor subtype, but not in tissues known to contain alpha1B-adrenoceptor (spleen, liver) and alpha1D-adrenoceptor (aorta). Scatchard analysis of [125I]L-762,459 binding in rat cerebral cortex and prostate indicated a single binding site with a Kd of 0.7 nM and Bmax of 11 (cerebral cortex) and 1 (prostate) pmole/g tissue. Specific and saturable [125I]L-762,459 binding was also found in human cerebral cortex, liver, prostate and vas deferens (Kd = 0.2-0.4 nM, Bmax = 0.4-4 pmole/g tissue). The specific binding in rat and human tissues was competed by non-selective alpha1-adrenoceptor compounds (Ki values in nM: prazosin (0.14-1.2), terazosin (1.8-5.9) and phentolamine (2.4-11)) and selective alpha1A-adrenoceptor compounds [Ki values in nM: (+) niguldipine (0.04-1.2) and SNAP 5399 ((+/-)-2-((2-aminoethyl)oxy)methyl-5-carboxamido-6-ethyl-4-(4-nitropheny l)-3-N-(3-(4,4-diphenylpiperidin-1-yl)propyl)carboxamido-1,4-dihyd ropyridine hydrate (0.5-4.8)]. The results were consistent with the selective binding of [125I]L-762,459 to the alpha1A-adrenoceptor. The specific labeling of the alpha1A-adrenoceptor subtype by [125I]L-762,459 may make it a useful tool to localize the distribution of the alpha1A-adrenoceptor.

Radioiodinated endothelin-1: a radiotracer for imaging endothelin receptor distribution and occupancy.

Endothelin (ET) is one of the most potent vasoconstrictors known. Recently, ET has been implicated in various diseases, e.g., acute renal failure and congestive heart failure, which present the possibility of treating such diseases with endothelin receptor antagonists. However, establishing the dosages for these antagonists may be difficult because no convenient physiologic indicator of action exists, and because of complexities in receptor function. Two receptor subtypes have been identified for which selective antagonists have been reported (e.g., BQ-123 for the ETA receptor and BQ-788 for the ETB receptor). Of the three natural peptide hormones (ET-1, ET-2, and ET-3), ET-1 exhibits high affinity for both subtypes of receptor. Using the selective peptide antagonists, and a nonpeptide antagonist with relatively balanced affinity for the two subtypes (L-749,329), we have characterized the interactions of [125I]ET-1 with its receptors in vivo (in rat). BQ-123, BQ-788, and L-749,329 inhibited binding consistent with binding to a single receptor site. However, the sum of inhibition by the selective antagonist was greater than 100% (as defined by inhibition with L-749,329), which suggests (a) lower in vivo selectivity than determined in vitro and/or (b) receptor subtype interactions. The latter explanation is supported, in part, by in vitro autoradiographic studies as well as studies in isolated tissues and cells. We synthesized ET-1 labeled with I-123 and obtained images of receptor distribution in both rat and rhesus monkey and have demonstrated our ability to visualize, via planar, noninvasive imaging, the occupancy of endothelin receptor by antagonists in both kidney and lung. [123I]ET-1 can therefore be used to determine clinical dosages of antagonist needed for receptor saturation.

Receptor binding radiotracers for the angiotensin II receptor: radioiodinated [Sar1, Ile8]Angiotensin II.

The potential for imaging the angiotensin II receptor was evaluated using the radioiodinated peptide antagonist [125I][Sar1, Ile8)angiotensin II. The radioligand provides a receptor-mediated signal in several tissues in rat (kidneys, adrenal and liver). The receptor-mediated signal of 3% ID/g kidney cortex should be sufficient to permit imaging, at least via SPECT. The radiotracer is sensitive to reductions in receptor concentration and can be used to define in vivo dose-occupancy curves of angiotensin II receptor ligands. Receptor-mediated images of [123I][Sar1, Ile8]angiotensin II were obtained in the rat kidney and Rhesus monkey liver.

Characterization of the binding of [125I]L-735,286: a new nonpeptide angiotensin II AT1 receptor radioligand.

[125I]L-735,286, a new potent and AT1-selective nonpeptide angiotensin II receptor radioligand, bound saturably to whole adrenal membranes. Scatchard and Hill plot analysis indicates a single class of high affinity (Kd = 0.5 nM) binding sites. The potencies of various angiotensin II agonists and antagonists in displacing specific [125I]L-735,286 binding are in good agreement with their potencies in displacing the binding of [125I]Sar1,Ile8-AII to adrenal AT1 receptors. The AT2 selective ligand, PD121981 had no effect on specific [125I]L-735,286 binding. In autoradiographic studies using rat kidney slices, specific labeling of [125I]L-735,286 was abolished by coincubation with saralasin. Collectively, the data indicated that [125I]L-735,286 represents a new, potent nonpeptide antagonist radioligand suitable for the study of angiotensin II AT1 receptors.

[125I]PIP HOE 140, a high affinity radioligand for bradykinin B2 receptors.

A high affinity radioligand for bradykinin B2 receptors was prepared by coupling an activated ester of [125I]4-iodobenzoic acid to the amino terminus nitrogen of the potent B2 antagonist HOE 140. The ligand, [125I]para-iodophenyl HOE 140 ([125I]PIP HOE 140), bound to a homogeneous set of sites in guinea pig ileal membranes with an equilibrium dissociation constant of 15 pM and a maximal binding density of 193 fmole/mg protein. Competition studies with a number of BK-related peptides indicated that the ligand specifically labeled B2 receptors in the preparation. The results suggest that [125I]PIP HOE 140 will be a useful tool for future studies of B2 receptors.

(+)-3-[123I]Iodo-MK-801: synthesis and characterization of binding to the N-methyl-D-aspartate receptor complex.

Synthetic methods have been established for preparing high specific activity (+)-3-[123I]Iodo-MK-801 in high radiochemical yield. The binding of the radiotracer to rat cortical membranes has been examined to assess its potential use as an in vivo imaging agent for the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. Under the conditions of the assay, specific (+)-3-[123I]Iodo-MK-801 binding to membrane homogenates represented greater than 95% of the total binding. Several structurally distinct, noncompetitive NMDA receptor antagonists inhibited binding with potencies in accordance with their reported inhibitory activity at the receptor complex. The concentration of (+/-)-3-Iodo-MK-801 required to inhibit 50% of (+)-3-[123I]Iodo-MK-801 binding (IC50) was 3.4 nM when using a low ionic strength assay buffer and 5.5 nM in a physiological buffer. In a thoroughly washed membrane preparation, (+)-3-[123I]Iodo-MK-801 binding was enhanced by L-glutamate and glycine at concentrations known to activate the NMDA receptor. The results indicate that (+)-3-[123I]Iodo-MK-801 specifically labels the NMDA receptor complex in rat brain membranes and the retention of high affinity under near physiological assay conditions suggests that it may be useful as a SPECT imaging agent for the receptor in vivo.

Improved synthesis of N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid and analogs.

A new synthesis of N-(2,6-dimethylphenylcarbamoyl-methyl)iminodiacetic acid directly from nitrilotriacetic acid was developed. Six analogs also were synthesized. Their technetium Tc 99m complexes were prepared and characterized. Electrophoresis and chromatography were used to determine the radiochemical purity of each complex.

A simple radiometric in vitro assay for acetylcholinesterase inhibitors.

A radiometric method for screening acetylcholinesterase inhibitors has been described. The method is based on the production of [14C]carbon dioxide from the hydrolysis of acetylcholine. The inhibitory concentration at 50% (IC50) values for several known acetylcholinesterase inhibitors were in agreement with literature values. The new radiometric method is simple, inexpensive, and has the potential for automation.

Electrophoretic determination of charge on carrier-free 99mTc-labeled complexes.

A new method for determining the charge on carrier-free 99mTc-labeled complexes is described. This method and mixed-ligand experiments were used to determine if the charge on the technetium 99m complex of N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetic acid (I) is - 1 and if the ligand to technetium ratio in the complex is 2:1. The preparation of an iodinated analog (II) of I and its 99mTc-labeled complex is described, as is the biodistribution of the 99mTc-labeled complex in mice. The synthesis of the 51Cr(III)- and 57Co(III)-labeled complexes also are described. The net biliary excretion in mice of both the 99mTc- and 51Cr-labeled complexes of II was significantly greater than that of the 99mTc-labeled complex of I.